Carbamyl phosphate synthetase I is a mitochondrial enzyme in mammalian cells that supplies carbamyl phosphate for arginine and urea biosynthesis. In procaryotes and yeast, the arginine-specific carbamyl phosphate synthetase in localized in the cytoplasm. The catalytic and physical properties of the mammalian enzyme suggest that it has evolved as a result of a fusion of the genes coding for two separate subunits of the procaryotic and fungal enzymes. The objective of the studies outlined in the present project is to clone the genes for the two subunits of the yeast carbamyl phosphate synthetase and the single gene of the rat liver enzyme. Their gene sequences will be determined as will the sequences of the E. coli genes which have already been cloned. A comparison of the DNA and protein sequences of the three arginine-specific carbamyl phosphate synthetases, from E. coli, yeast, and rat, should provide information about the evolution of this important metabolic enzyme.